This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Molecular oxygen is a fundamental requirement for aerobic life, yet oxidative stress damages macromolecules and can be lethal if unchecked. Thus far, allosteric conformational changes that regulate transcription in response to the redox state have been visualized in few, if any cases. The long term goal of the proposed research is to elucidate novel and essential allosteric mechanisms that regulate DNA-binding in response to the intracellular redox state. In Gram-positive bacteria, the redox-sensing repressor (Rex) adopts the elegant approach of distinguishing the reduced and oxidized states of the essential cofactor, NAD. Rex binds the DNA target and represses transcription of respiratory genes in the presence of oxidized NAD+, yet authorizes transcription when reduced NADH levels abnormally rise. This laboratory previously determined the X-ray structure of the Rex/NADH complex. Although the Rex/NADH structure provided one important view of the state of Rex that is unable to recognize DNA, the fundamental mechanism of the allosteric response of Rex to NADH/NAD+ remains unknown. The structure of the Rex/NAD+/DNA complex is needed to understand how Rex rearranges to recognize DNA, and how the slight differences between NAD+/NADH trigger this transition. Towards this goal, Rex/NAD+/DNA cocrystals have been obtained. The presence of both DNA and protein was verified from the A280:A260 ratio of dissolved crystals. Although the crystals diffract beyond 3 resolution, a 300 c-axis limits our ability to collect complete, high-resolution data in-house. The structure will be solved by MIR or MAD phasing with brominated-DNA, and additional phase information provided by a single SeMet per monomer. Based on the incompatible shapes of Rex/NADH and DNA, Rex is likely to undergo a large conformational change following NADH/NAD+ exchange. The hypothesized conformational rearrangement of Rex would reveal a new paradigm for transcriptional regulation in response to the minimal differences between NAD+ and NADH.
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